Peter J. Griffiths

Dilated and Hypertrophic Cardiomyopathy Mutations in Troponin and α-Tropomyosin Have Opposing Effects on the Calcium Affinity of Cardiac Thin Filaments

Dilated cardiomyopathy and hypertrophic cardiomyopathy (HCM) can be caused by mutations in thin filament regulatory proteins of the contractile apparatus. In vitro functional assays show that, in general, the presence of dilated cardiomyopathy mutations decreases the Ca2+ sensitivity of contractility, whereas HCM mutations increase it. To assess whether this functional phenomenon was a direct result of altered Ca2+ affinity or was caused by altered troponin–tropomyosin switching, we assessed Ca2+ binding of the regulatory site of cardiac troponin C in wild-type or mutant troponin complex and thin filaments using a fluorescent probe (2-[4′-{iodoacetamido}aniline]-naphthalene-6-sulfonate) attached to Cys35 of cardiac troponin C. The Ca2+-binding affinity (pCa50=6.57±0.03) of reconstituted troponin complex was unaffected by all of the HCM and dilated cardiomyopathy troponin mutants tested, with the exception of the troponin I Arg145Gly HCM mutation, which caused an increase (&Dgr;pCa50=+0.31±0.05). However, when incorporated into regulated thin filaments, all but 1 of the 10 troponin and &agr;-tropomyosin mutants altered Ca2+-binding affinity. Both HCM mutations increased Ca2+ affinity (&Dgr;pCa50=+0.41±0.02 and +0.51±0.01), whereas the dilated cardiomyopathy mutations decreased affinity (&Dgr;pCa50=−0.12±0.04 to −0.54±0.04), which correlates with our previous functional in vitro assays. The exception was the troponin T Asp270Asn mutant, which caused a significant decrease in cooperativity. Because troponin is the major Ca2+ buffer in the cardiomyocyte sarcoplasm, we suggest that Ca2+ affinity changes caused by cardiomyopathy mutant proteins may directly affect the Ca2+ transient and hence Ca2+-sensitive disease state remodeling pathways in vivo. This represents a novel mechanism for this class of mutation.

Stiffness and force in activated frog skeletal muscle fibers

Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs. A set of equations is given in the Appendix that describes the frequency dependence of the applied sinusoid and its response. This model predicts that frequency dependent changes can be used as a measure of a change in stiffness.

An examination of the ability ofinositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea

We have examined the ability of inositol 1,4,5‐trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10–500 μM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impenneant anion by Cl−, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium‐induced calcium release. Free Mg2+ was lowered to 40 μM and 15 mM D‐2,3‐diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0–0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadmium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4° to 22°C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3‐induced calcium release from rat liver microsomes, had no effect at 50 μM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation‐contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.

Cross-bridge attachment and stiffness during isotonic shortening of intact single muscle fibers

Equatorial x-ray diffraction pattern intensities (I10 and I11), fiber stiffness and sarcomere length were measured in single, intact muscle fibers under isometric conditions and during constant velocity (ramp) shortening. At the velocity of unloaded shortening (Vmax) the I10 change accompanying activation was reduced to 50.8% of its isometric value, I11 reduced to 60.7%. If the roughly linear relation between numbers of attached bridges and equatorial signals in the isometric state also applies during shortening, this would predict 51-61% attachment. Stiffness (measured using 4 kHz sinusoidal length oscillations), another putative measure of bridge attachment, was 30% of its isometric value at Vmax. When small step length changes were applied to the preparation (such as used for construction of T1 curves), no equatorial intensity changes could be detected with our present time resolution (5 ms). Therefore, unlike the isometric situation, stiffness and equatorial signals obtained during ramp shortening are not in agreement. This may be a result of a changed crossbridge spatial orientation during shortening, a different average stiffness per attached crossbridge, or a higher proportion of single headed crossbridges during shortening.

Changes in myosin S1 orientation and force induced by a temperature increase

Force generation in myosin-based motile systems is thought to result from an angular displacement of the myosin subfragment 1 (S1) tail domain with respect to the actin filament axis. In muscle, raised temperature increases the force generated by S1, implying a greater change in tail domain angular displacement. We used time-resolved x-ray diffraction to investigate the structural corollary of this force increase by measuring M3 meridional reflection intensity during sinusoidal length oscillations. This technique allows definition of S1 orientation with respect to the myofilament axis. M3 intensity changes were approximately sinusoid at low temperatures but became increasingly distorted as temperature was elevated, with the formation of a double intensity peak at maximum shortening. This increased distortion could be accounted for by assuming a shift in orientation of the tail domain of actin-bound S1 toward the orientation at which M3 intensity is maximal, which is consistent with a tail domain rotation model of force generation in which the tail approaches a more perpendicular projection from the thin filament axis at higher temperatures. In power stroke simulations, the angle between S1 tail mean position during oscillations and the position at maximum intensity decreased by 4.7°, corresponding to a mean tail displacement toward the perpendicular of 0.73 nm for a temperature-induced force increase of 0.28 P0 from 4 to 22°C. Our findings suggest that at least 62% of crossbridge compliance is associated with the tail domain.

Volume Changes of the Myosin Lattice Resulting from Repetitive Stimulation of Single Muscle Fibers

Single muscle fibers at 1 degreesC were subjected to brief tetani (20 Hz) at intervals of between 20 s and 300 s over a period of up to 2 h. A band lattice spacing increased during this period at a rate inversely dependent on the rest interval between tetani. Spacing increased rapidly during the first 10 tetani at a rate equivalent to the production of 0.04 mOsmol.liter-1 of osmolyte per contraction, then continued to expand at a much slower rate. For short rest intervals, where lattice expansion was largest, spacing increased to a limiting value between 46 and 47 nm (sarcomere length 2.2 micrometer), corresponding to accumulation of 30 mOsmol.liter-1 of osmolytes, where it remained constant until repetitive stimulation was terminated. At this limiting spacing, force was reduced by up to 30%. The effect of lattice swelling on the lattice compression that accompanies isometric force recovery from unloaded shortening was to increase the compression, similar to that observed in hypotonic media at a similar spacing. During recovery from repetitive stimulation, spacing recompressed to its original value with a half-time of 15-30 min. These findings suggest that mechanical activity produces an increase in osmotic pressure within the cell as a result of product accumulation from cross-bridge and sarcoplasmic reticulum ATPases and glycolysis.

Frequency-dependent distortion of meridional intensity changes during sinusoidal length oscillations of activated skeletal muscle.

Bundles of intact, tetanized skeletal muscle fibers from Rana temporaria were subjected to sinusoidal length oscillations in the frequency domain 100 Hz to 3 kHz while measuring force and sarcomere length. Simultaneously, intensity of the third-order x-ray reflection of the axial myosin unit cell (I(M3)) was measured using synchrotron radiation. At oscillation frequencies <1 kHz, I(M3) was distorted during the shortening phase of the sinusoid (i.e., where bundle length was less than rest length). Otherwise, during the stretch phase of oscillations at all frequencies, during the shortening phase of oscillations above 1 kHz, and for bundles in the rigor state, I(M3) was approximately sinusoidal in form. Mean I(M3) during oscillations was reduced by 20% compared to the isometric value, suggesting a possible change in S1 disposition during oscillations. However, the amplitude of length change required to produce distortion (estimated from the phase angle at which distortion was first evident) corresponded to that of a step release sufficient to reach the maximum I(M3), indicating a mean S1 disposition during oscillations close to that during an isometric tetanus. The mechanical properties of the bundle during oscillations were also consistent with an unaltered S1 disposition during oscillations.

Fluorescence changes from single striated muscle fibres injected with labelled troponin C (TnCDANZ)

A fluorescently labelled derivative of the calcium binding subunit of troponin, TnC, has been injected into isolated striated muscle fibres from the barnacle Balanus nubilus. The Ca2+ affinity of isolated TnC is close to that of intact troponin when located in the thin filament. Excitation of the TnCDANZ within the muscle cell (325nm) revealed a marked fluorescence at 510 nm and was similar to that observed in vitro, which was absent at 400 or 600 nm after subtraction of the fibre autofluorescence. High Ca2+ salines increased the fluorescence at 510 nm by roughly 2 times. Single voltage clamp pulses produced a rapid rise in fluorescence at 510 nm after allowing for any non‐specific changes at 400 nm, and this signal preceded force development by approx. 55 ms at 22° C. It reached a maximum at the same time as force and subsequently decayed more slowly. The fluorescence signal increased in magnitude with increase in stimulus intensity. These results suggest that Ca2+ attaches rapidly to the contractile filament, but is lost relatively slowly and imply a slow decay of the activation process.

Effects of solution tonicity on crossbridge properties and myosin lever arm disposition in intact frog muscle fibres

The aims of this study were to investigate the effects of solution tonicity on muscle properties, and to verify their consistence with the lever arm theory of force generation. Experiments were made in single muscle fibres and in fibre bundles from the frog, using both fast stretches and time‐resolved X‐ray diffraction, in isotonic Ringer solution (1T), hypertonic (1.4T) and hypotonic (0.8T) solutions. Fast stretches (0.4–0.6 ms duration and 16–25 nm per half‐sarcomere (nm hs−1) amplitude) were applied at various tensions during the force development in isometric tetani. Force increased during the stretch up to a peak (critical tension, Pc) at which it started to fall, in spite of continued stretching. In all solutions, Pc was proportional to the initial isometric tension developed. For a given isometric tension, Pc increased with solution tonicity and occurred at a precise sarcomere elongation (critical length, Lc) which also increased with tonicity. M3 meridional layer line intensity (IM3) was measured during the application of sinusoidal length oscillations (1 kHz frequency, and about 2% fibre length amplitude) at tetanus plateau. IM3 changed during the length oscillations in a sinusoidal manner in phase opposition to length changes, but a double peak distortion occurred at the peak of the release phase. The presence of the distortion, which decreased with tonicity, allowed calculation of the mean position of the myosin head (S1) during the oscillation cycle. In agreement with the lever arm theory, both X‐ray diffraction and mechanical data show that solution tonicity affects S1 mean position and consequently crossbridge individual extension and force, with no effect on crossbridge number. The force needed to break the single crossbridge was insensitive to solution tonicity suggesting a non‐ionic nature of the actomyosin bond.

Mechanical characteristics of skinned and intact muscle fibres from the giant barnacle, Balanus nubilus

Intact muscle fibres fromBalanus nubilus develop tensions of up to 600 kN sd m−2 during electrical stimulation. The rise of tension occurs with a half-time (177 ms at 12° C) about fivefold longer than that of tetanised frog muscle at the same temperature. The response of myofibrillar bundles to a rapid stretch resembles that of frog muscle but has a yo value (i.e. the size of an instantaneous release necessary to just discharge tension) which is ca. 2.5 times smaller, and phase 2 of the tension transient (the “quick phase”) occurs at a rate comparable to that of frog muscle. In contrast, the ATPase activity (0.018 mmoles · kg wet weight−1 · s−1) of this preparation and its maximum shortening velocity (0.15–0.16 muscle lengths · s−1) are both at least fivefold slower than frog muscle. These findings can be accounted for by a cross-bridge cycle in barnacle muscle in which events prior and subsequent to the tension generating step(s) occur at a rate at least fivefold slower than comparable steps in frog muscle, but the step(s) associated with tension development occur at similar rates in the two preparations. Since the rate of mechanical relaxation in barnacle muscle is modified in the presence of intracellular calcium buffers and by depolarisation-induced elevation of the free calcium during the relaxation phase, it is proposed that the time course of relaxation is not determined exclusively by the kinetics of the cross-bridge cycle, but is also dependent on the free calcium concentration during relaxation.